The analysis of metabolites in rainbow trout white muscle: a comparison of different sampling and processing methods
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abstract
We have investigated the effects of different sampling and processing methods on metabolite concentrations [glycogen (Gly), glucose (Glu), lactate (Lac), pyruvate (Pyr), ammonia (Amm), creatine phosphate (PCr), creatine (Cr), and adenosine triphosphate (ATP)] measured in white muscle of rainbow trout at rest and immediately after exhaustive exercise. When samples were taken from resting fish by rapid needle biopsy (without anaesthesia), direct freezing of the needles in liquid N2 yielded lower Lac and Glu levels than if the muscle cores were quickly blown out into liquid N2. However, killing of the fish by an overdose of MS‐222 followed by freeze‐clamping of excised muscle was superior to the biopsy method in preserving high levels of PCr and Gly (91 and 62% higher, respectively). In parallel, the MS‐222 method also yielded lower levels of Amm (80%) and Lac (47%). Samples freeze‐clamped by the MS‐222 method were used to evaluate three methods of subsequent processing for enzymatic analysis of metabolites: classic glass homogenization (GH) in 8% perchloric acid (PCA) c. mortar and pestle (MP) pulverization or freeze‐drying (FD) prior to PCA extraction. For all metabolites, GH and MP methods produced similar values. However, the FD technique yielded 20% higher PCr levels which represented over 80% phosphorylation of the total Cr pool at rest, the highest ever reported via enzymatic analysis. Glu was also higher by FD, bul Gly, Lac, and ATP were not affected. Indeed ATP was relatively stable throughout all sampling and processing procedures. MP, GH, MP&GH combination, and high speed motor driven grinding techniques all yielded similar Amm levels in resting muscle. However, tests demonstrated that even brief thawing of tissue greatly elevated Amm, while FD resulted in artificially low Amm values due to evaporative losses during lyophilization. Overall, muscle sampling by freeze‐clamping on trout killed by MS‐222 overdose, followed by FD prior to PCA extraction, appears to be the best combination for the measurement of all white muscle metabolites except Amm, for which MP or GH are preferable.
